Antisense modulation of fan expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of FAN. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding FAN. Methods of using these compounds for modulation of FAN expression and for treatment of diseases associated with expression of FAN are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulating the expression of FAN. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human FAN. Such oligonucleotides have been shown to modulate the expression of FAN.

BACKGROUND OF THE INVENTION

One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals into intracellular signals that in turn modulate biochemical pathways. Examples of such extracellular signaling molecules include growth factors, cytokines, and chemokines. The cell surface receptors of these molecules and their associated signal transduction pathways are therefore among the principal means by which cellular behavior is regulated. Because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of signal transduction pathways. Consequently, considerable attention has been devoted to the characterization of these proteins.

Tumor necrosis factor (TNF), a cytokine produced by activated macrophages, initiates a variety of cellular responses. These responses include proliferation, differentiation and activation as well as apoptosis, inflammatory and immunoregulatory responses, and all are mediated through one of two receptors, TNF-R55 or TNF-R75 (Adam-Klages et al., Cell, 1996, 86, 937-947).

Unlike most receptors, TNF-R55 has no intrinsic enzymatic activity and therefore relays signals through protein binding motifs on its cytoplasmic domain. One such domain is known as the death domain and regulates binding of proteins involved in the signaling pathway leading to apoptosis. Another recently discovered binding domain is the the NSD (N-SMase activating domain). This domain comprises the binding site for a newly discovered protein, FAN.

FAN (factor associated with N-SMase activation), a regulatory protein of the WD-repeat protein family, was identified as a mediator of TNF-induced activation of N-SMase and likely plays a role in the inflammation process (Adam et al., J. Inflamm., 1995, 47, 61-66; Adam-Klages et al., Cell, 1996, 86, 937-947). Binding of TNF to the extracellular side of the receptor initiates a pathway on the intracellular side in which FAN activates the enzyme N-SMase (neutral sphingomyelinase). This enzyme, in turn, hydrolyzes sphingomyelin to release ceramide, an important second messenger, which then accumulates at the plasma membrane. This accumulation activates proline-directed protein kinases, which then activate the cytosolic phospholipase A2 producing arachidonic acid resulting in the generation of proinflammatory metabolites such as prostaglandins and leukotrienes (Adam-Klages et al., J. Leukoc. Biol., 1998, 63, 678-682).

Currently, there are no known therapeutic agents which effectively inhibit the synthesis of FAN. To date, strategies aimed at inhibiting FAN function have involved the use of peptide-hydroxamate metalloproteinase inhibitors to block the proteolytic processing of TNF-R55 and TNF-R75(Gallea-Robache et al., Cytokine, 1997, 9, 340-346). However, these targeting strategies are not specific to FAN and could affect all of the signaling pathways downstream of the TNF-R55 receptor. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting FAN function.

Antisense oligonucleotides capable of inhibiting protein expression or function may prove useful in a number of therapeutic and research applications and could thereby provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding FAN, and which modulate the expression of FAN. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of FAN in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of FAN by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding FAN, ultimately modulating the amount of FAN produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding FAN. As used herein, the terms "target nucleic acid" and "nucleic acid encoding FAN" encompass DNA encoding FAN, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense". The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of FAN. In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding FAN. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding FAN, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.

The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an MRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'--5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.

Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂ --N(CH₃)--O--CH₂ -- known as a methylene (methylimino) or MMI backbone!, --CH₂ --O--N(CH₃)--CH₂ --, --CH₂ -- N(CH₃)--N(CH₃)--CH₂ -- and --O--N(CH₃)--CH₂ --CH₂ -- wherein the native phosphodiester backbone is represented as --O--P--O--CH₂ --! of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O (CH₂)_(n) O!_(m) CH₃, O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂, and O(CH₂)_(n) ON (CH₂)_(n) CH₃)!₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH₂ CH₂ OCH₃, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE, as described in U.S. patent application Ser. No. 09/016,520, filed on Jan. 30, 1998, which is commonly owned with the instant application and the contents of which are herein incorporated by reference.

Other preferred modifications include 2'-methoxy (2'-O--CH₃), 2'-aminopropoxy (2'-OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference and allowed U.S. patent application Ser. No. 08/468,037, filed on Jun. 5, 1995, U.S. Pat. No. X,XXX,XXX, which is commonly owned with the instant application and is also herein incorporated by reference.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. No. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and allowed U.S. patent application Ser. No. 08/465,880, filed on Jun. 6, 1995, which is commonly owned with the instant application and also herein incorporated by reference.

The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE (S-acetyl-2-thioethyl) phosphate! derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of FAN is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding FAN, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding FAN can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of FAN in a sample may also be prepared.

The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and/or formulations comprising the oligonucleotides of the present invention may also include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included. Penetration enhancers are described in pending U.S. patent application Ser. No. 08/886,829, filed on Jul. 1, 1997, and pending U.S. patent application Ser. No. 08/961,469, filed on Oct. 31, 1997, both of which are commonly owned with the instant application and both of which are herein incorporated by reference.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid, lyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8: 2, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1, 1-33; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presently preferred fatty acids are sodium caprate and sodium laurate, used singly or in combination at concentrations of 0.5 to 5%.

Preferred penetration enhancers are disclosed in pending U.S. patent application Ser. No. 08/886,829, filed on Jul. 1, 1997, which is commonly owned with the instant application and which is herein incorporated by reference.

The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Preferred bile salts are described in pending U.S. patent application Ser. No. 08/886,829, filed on Jul. 1, 1997, which is commonly owned with the instant application and which is herein incorporated by reference. A presently preferred bile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St. Louis, Mo.), generally used at concentrations of 0.5 to 2%.

Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations. Preferred combinations include CDCA combined with sodium caprate or sodium laurate (generally 0.5 to 5%).

Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8: 2, 92-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8: 2, 92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8: 2, 92-191); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

As used herein, "carrier compound" refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioated oligonucleotide in hepatic tissue is reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

In contrast to a carrier compound, a "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.

Regardless of the method by which the antisense compounds of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the compounds and/or to target the compounds to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layer(s) made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech., 1995, 6, 698-708).

Liposome preparation is described in pending U.S. patent application Ser. No. 08/961,469, filed on Oct. 31, 1997, which is commonly owned with the instant application and which is herein incorporated by reference.

Certain embodiments of the invention provide for liposomes and other compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 1206-1228). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Examples of antisense oligonucleotides include, but are not limited to, those directed to the following targets as disclosed in the indicated U.S. Patents, or pending U.S. applications, which are commonly owned with the instant application and are hereby incorporated by reference, or the indicated published PCT applications: raf (WO 96/39415, WO 95/32987 and U.S. Pat. Nos. 5,563,255, issued Oct. 8, 1996, and 5,656,612, issued Aug. 12, 1997), the p120 nucleolar antigen (WO 93/17125 and U.S. Pat. No. 5,656,743, issued Aug. 12, 1997), protein kinase C (WO 95/02069, WO 95/03833 and WO 93/19203), multidrug resistance-associated protein (WO 95/10938 and U.S. Pat. No. 5,510,239, issued Mar. 23, 1996), subunits of transcription factor AP-1 (pending application U.S. Ser. No. 08/837,201, filed Apr. 14, 1997), Jun kinases (pending application U.S. Ser. No. 08/910,629, filed Aug. 13, 1997), MDR-1 (multidrug resistance glycoprotein; pending application U.S. Ser. No. 08/731,199, filed Sep. 30, 1997), HIV (U.S. Pat. Nos. 5,166,195, issued Nov. 24, 1992 and 5,591,600, issued Jan. 7, 1997), herpesvirus (U.S. Pat. No. 5,248,670, issued Sep. 28, 1993 and U.S. Pat. No. 5,514,577, issued May 7, 1996), cytomegalovirus (U.S. Pat. Nos. 5,442,049, issued Aug. 15, 1995 and 5,591,720, issued Jan. 7, 1997), papillomavirus (U.S. Pat. No. 5,457,189, issued Oct. 10, 1995), intercellular adhesion molecule-1 (ICAM-1) (U.S. Pat. No. 5,514,788, issued May 7, 1996), 5-lipoxygenase (U.S. Pat. No. 5,530,114, issued Jun. 25, 1996) and influenza virus (U.S. Pat. No. 5,580,767, issued Dec. 3, 1996). Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀ s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis

Deoxy and 2'-alkoxy amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203! using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

2'-Fluoro amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previously Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841! and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2'-alpha-fluoro atom is introduced by a S_(N) 2-displacement of a 2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5'-DMT- and 5'-DMT-3'-phosphoramidites.

2' -Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by the modification of a literature procedure in which 2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-2'-fluorocytidine was synthesized via amination of 2'-deoxy-2'-fluorouridine, followed by selective protection to give N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

2,2'-Anhydro 1-(beta-D-arabinofuranosyl)-5-methyluridine!

5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂ Cl₂ /Acetone/MeOH (20:5:3) containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃ CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂ SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/Pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1 L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L) Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

2'-(Aminooxyethyl) nucleoside amidites and 2'-(dimethylaminooxyethyl) nucleoside amidites

Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per the methods of U.S. patent applications Ser. No. 10/037,143, filed Feb. 14, 1998, and Ser. No. 09/016,520, filed Jan. 30, 1998, each of which is commonly owned with the instant application and is herein incorporated by reference.

Example 2

Oligonucleotide synthesis

Unsubstituted and substituted phosphodiester (P=O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P=S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 hr), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P=O or P=S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".

2'-O-Me!-- 2'-deoxy!-- 2'-O-Me! Chimeric

Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2'-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 Ammonia/Ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2' positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

2'-O-(2-Methoxyethyl)!-- 2'-deoxy!-- 2'-O-(Methoxyethyl)! Chimeric Phosphorothioate

Oligonucleotides

2'-O-(2-methoxyethyl)!-- 2'-deoxy!-- -2'-O-(methoxyethyl)! chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

2'-O-(2-Methoxyethyl)Phosphodiester!-- 2'-deoxy Phosphorothioate!-- 21-O-(2-Methoxyethyl) Phosphodiester! Chimeric Oligonucleotides

2'-O-(2-methoxyethyl phosphodiester!-- 2'-deoxy phosphorothioate!-- 2'-O-(methoxyethyl) phosphodiester! chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹ P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected betacyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected with concentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

Cell culture and oligonucleotide treatment

The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following four cell types are provided for illustrative purposes, but other cell types can be routinely used.

T-24 cells:

The transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

Treatment with antisense compounds:

When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired oligonucleotide at a final concentration of 150 nM. After 4 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Analysis of oligonucleotide inhibition of FAN expression

Antisense modulation of FAN expression can be assayed in a variety of ways known in the art. For example, FAN mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. Other methods of PCR are also known in the art.

FAN protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to FAN can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

Poly(A)+mRNA isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pippeting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 ∥L water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

Example 13

Real-time Quantitative PCR Analysis of FAN mRNA Levels

Quantitation of FAN mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension). FAN probes and primers were designed to hybridize to the human FAN sequence, using published sequence information (GenBank accession number X96586, incorporated herein as SEQ ID NO: 1).

For FAN the PCR primers were:

forward primer: CACACTGCTGAGAGCTACATGCT (SEQ ID NO: 2)

reverse primer: GTTGTTGAGGTGAAGGAGGTACTGAT (SEQ ID NO: 3) and

the PCR probe was: FAM-CAGTGGCAGCGTGGACACCTTTCC-TAMRA (SEQ ID NO: 4) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 5)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)and the

PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 7) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

Northern blot analysis of FAN mRNA levels

Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions with a FAN specific probe prepared by PCR using the forward primer CACACTGCTGAGAGCTACATGCT (SEQ ID NO: 2) and the reverse primer GTTGTTGAGGTGAAGGAGGTACTGAT (SEQ ID NO: 3). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

Antisense inhibition of FAN expression-phosphorothioate oligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human FAN RNA, using published sequences (GenBank accession number X96586, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X96586), to which the oligonucleotide binds. All compounds in Table 1 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on FAN mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, "N.D." indicates "no data".

                  TABLE 1     ______________________________________     Inhibition of FAN mRNA levels by phosphorothioate     oligodeoxynucleotides                                        %                   TARGET               Inhi- SEQ ID     ISIS #           REGION  SITE    SEQUENCE     bition                                              NO.     ______________________________________     24481 Coding  57      aatctctccttggagtag                                        39     8     24482 Coding    109    ggctctatgctgttcaaa                                              61                                                      9     24483 Coding    148    tttcctttcatggtgact                                              57                                                    10     24484 Coding    213    ggctgggatattgaatct                                              28                                                    11     24485 Coding    243    atacagtctctcaaagga                                              27                                                    12     24486 Coding    291    tttgtgaagtgtctattg                                              49                                                    13     24487 Coding    336    aaatatacctgactgaaa                                                     14     24488 Coding    379    cctttctattttatatgg                                              26                                                    15     24489 Coding    441    gtctccacaacatcttcc                                              14                                                    16     24490 Coding    474    tcaaggcaggatgctctg                                              77                                                    17     24491 Coding    522    cgagactgcaaaatagct                                              68                                                    18     24492 Coding    586    ttccatgtgcagcttttc                                              66                                                    19     24493 Coding   649     tgtgtccgtgatgcacac                                              76                                                    20     24494 Coding    706    gagtgttatctggaccac                                              72                                                    21     24495 Coding    742    gccgtgcctccttttgta                                              53                                                    22     24496 Coding    807    tttaggtagatgtcggaa                                              48                                                    23     24497 Coding    865    ctctaggtatgtggcaat                                                     24     24498 Coding    907    ctgcagcatgtagctctc                                              17                                                    25     24499 Coding    960    aggttgttgaggtgaagg                                                     26     24500  Coding  1006   tggaaacacagggtactg                                              63                                                    27     24501 Coding   1071   agatcccggaaggttcct                                              78                                                    28     24502 Coding   1123   tgtcagtagtctctccag                                              75                                                    29     24503 Coding   1166   gactcccatacatgaact                                              49                                                    30     24504 Coding   1230   cacagcatatactctggt                                              65                                                    31     24505 Coding   1274    tgttgaacattctatctg                                              21                                                     32     24506 Coding   1323    ttaaaatccgttgcacca                                              45                                                     33     24507 Coding   1381    cttcaggctattgactag                                              54                                                     34     24508 Coding   1413    atctgtcctccttgtctc                                              53                                                     35     24509 Coding   1473    ctcttctggagaaagtcc                                              33                                                     36     24510 Coding   1520    actcgtgaaggtgttcag                                              66                                                     37     24511 Coding   1608    cctccttcataggtcagg                                              26                                                     38     24512  Coding  1655    gcatggctaccttctcat                                              85                                                     39     24513 Coding  1780     atctgccatagaagcatt                                              44                                                     40     24514 Coding   1905    gtgattccagtaactgct                                              64                                                     41     24515 Coding   2059    tatgacagtggcatctcc                                              35                                                     42     24516 Coding   2293    tacactgacatcatgttc                                              57                                                     43     24517 Coding   2539    gggctcatctgatgtcat                                                     44     24518 Coding   2653    tgtgtggccctgtattct                                              62                                                     45     24519 3'40  UTR                   2791     ttaaatagagttcaattt                                                     46     24520 3'40  UTR                   2947     cattttatctgccctaag                                              44                                                     47     ______________________________________

As shown in Table 1, SEQ ID NOs 8, 9, 10, 13, 17, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 45 and 47 demonstrated at least 30% inhibition of FAN expression in this assay and are therefore preferred.

Example 16

Antisense inhibition of FAN expression-phosphorothioate 2'-MOE gapmer oligonucleotides

In accordance with the present invention, a second series of oligonucleotides targeted to human FAN were synthesized. The oligonucleotide sequences are shown in Table 2. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X96586), to which the oligonucleotide binds.

All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data".

                  TABLE 2     ______________________________________     Inhibition of FAN mRNA levels by chimeric     phosphorothioate oligonucleotides having     2'40 -MOE wings and a deoxy gap                                        %                   TARGET               Inhi- SEQ ID     ISIS #           REGION  SITE        SEQUENCE        NO.         bition     ______________________________________     24521 Coding  57      aatctctccttggagtag                                        61     8     24522 Coding    109     ggctctatgctgttcaaa                                             77                                                      9     24523 Coding    148     tttcctttcatggtgact                                             67                                                     10     24524 Coding    213     ggctgggatattgaatct                                             69                                                     11     24525 Coding    243     atacagtctctcaaagga                                             60                                                     12     24526 Coding    291     tttgtgaagtgtctattg                                             69                                                     13     24527 Coding    336     aaatatacctgactgaaa                                             25                                                     14     24528 Coding    379     cctttctattttatatgg                                             29                                                     15     24529 Coding    441     gtctccacaacatcttcc                                             45                                                     16     24530 Coding    474     tcaaggcaggatgctctg                                             74                                                     17     24531 Coding    522     cgagactgcaaaatagct                                             87                                                     18     24532 Coding    586     ttccatgtgcagcttttc                                             77                                                     19     24533 Coding    649     tgtgtccgtgatgcacac                                             77                                                     20     24534 Coding    706     gagtgttatctggaccac                                             83                                                     21     24535 Coding    742     gccgtgcctccttttgta                                             64                                                     22     24536 Coding    807     tttaggtagatgtcggaa                                             73                                                     23     24537 Coding    865     ctctaggtatgtggcaat                                             54                                                     24     24538 Coding    907     ctgcagcatgtagctctc                                             66                                                     25     24539 Coding    960     aggttgttgaggtgaagg                                             65                                                     26     24540 Coding   1006    tggaaacacagggtactg                                             63                                                     27     24541 Coding   1071    agatcccggaaggttcct                                              0                                                      28     24542 Coding   1123    tgtcagtagtctctccag                                             76                                                     29     24543 Coding   1166    gactcccatacatgaact                                             67                                                     30     24544 Coding   1230    cacagcatatactctggt                                             78                                                     31     24545 Coding   1274    tgttgaacattctatctg                                              57                                                     32     24546 Coding  1323     ttaaaatccgttgcacca                                              52                                                     33     24547 Coding   1381    cttcaggctattgactag                                              60                                                     34     24548 Coding   1413    atctgtcctccttgtctc                                              57                                                     35     24549 Coding   1473    ctcttctggagaaagtcc                                              54                                                     36     24550 Coding   1520    actcgtgaaggtgttcag                                              77                                                     37     24551 Coding   1608    cctccttcataggtcagg                                              75                                                     38     24552 Coding   1655    gcatggctaccttctcat                                              77                                                     39     24553 Coding  1780     atctgccatagaagcatt                                                     40     24554 Coding   1905    gtgattccagtaactgct                                              57                                                     41     24555 Coding   2059    tatgacagtggcatctcc                                                     42     24556 Coding   2293    tacactgacatcatgttc                                              56                                                     43     24557 Coding   2539    gggctcatctgatgtcat                                              19                                                     44     24558 Coding   2653    tgtgtggccctgtattct                                              20                                                     45     24559 3'40  UTR                   2791     ttaaatagagttcaattt                                                     46     24560 3'40  UTR                   2947     cattttatctgccctaag                                              54                                                     47     ______________________________________

As shown in Table 2, SEQ ID NOs 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 43 and 47 demonstrated at least 40% inhibition of FAN expression in this experiment and are therefore preferred.

Example 17

Western blot analysis of FAN protein levels

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 hr after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to FAN is used, with a radiolabelled or fluorescently labeled secondary anitbody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

    __________________________________________________________________________     #             SEQUENCE LISTING     - <160> NUMBER OF SEQ ID NOS: 47     - <210> SEQ ID NO 1     <211> LENGTH: 3380     <212> TYPE: DNA     <213> ORGANISM: Homo sapiens     <220> FEATURE:     <221> NAME/KEY: CDS     <222> LOCATION: (13)..(2766)     - <400> SEQUENCE: 1     - gaattcgcct cg atg gcg ttt atc cgg aag aag cag - # cag gag cag cag       48                   Met Ala Ph - #e Ile Arg Lys Lys Gln Gln Glu Gln Gln     #             10     - ctg cag ctc tac tcc aag gag aga ttt tcc tt - #g ctg ctg ctt aac ttg       96     Leu Gln Leu Tyr Ser Lys Glu Arg Phe Ser Le - #u Leu Leu Leu Asn Leu     #         25     - gag gag tac tac ttt gaa cag cat aga gcc aa - #t cac att ttg cac aag      144     Glu Glu Tyr Tyr Phe Glu Gln His Arg Ala As - #n His Ile Leu His Lys     #     40     - ggc agt cac cat gaa agg aaa atc aga ggc tc - #c tta aaa ata tgt tca      192     Gly Ser His His Glu Arg Lys Ile Arg Gly Se - #r Leu Lys Ile Cys Ser     # 60     - aaa tcg gtg att ttt gaa cca gat tca ata tc - #c cag ccc atc atc aag      240     Lys Ser Val Ile Phe Glu Pro Asp Ser Ile Se - #r Gln Pro Ile Ile Lys     #                 75     - att cct ttg aga gac tgt ata aaa ata gga aa - #g cat gga gaa aat gga      288     Ile Pro Leu Arg Asp Cys Ile Lys Ile Gly Ly - #s His Gly Glu Asn Gly     #             90     - gcc aat aga cac ttc aca aag gca aaa tct gg - #g ggt att tca ctc att      336     Ala Asn Arg His Phe Thr Lys Ala Lys Ser Gl - #y Gly Ile Ser Leu Ile     #        105     - ttc agt cag gta tat ttc att aaa gaa cat aa - #t gtt gtt gca cca tat      384     Phe Ser Gln Val Tyr Phe Ile Lys Glu His As - #n Val Val Ala Pro Tyr     #   120     - aaa ata gaa agg ggc aaa atg gaa tat gtt tt - #t gaa ttg gat gtt ccc      432     Lys Ile Glu Arg Gly Lys Met Glu Tyr Val Ph - #e Glu Leu Asp Val Pro     125                 1 - #30                 1 - #35                 1 -     #40     - ggg aaa gtg gaa gat gtt gtg gag acg ttg ct - #t cag ctt cac aga gca      480     Gly Lys Val Glu Asp Val Val Glu Thr Leu Le - #u Gln Leu His Arg Ala     #               155     - tcc tgc ctt gac aaa ttg ggt gac caa acc gc - #c atg ata aca gct att      528     Ser Cys Leu Asp Lys Leu Gly Asp Gln Thr Al - #a Met Ile Thr Ala Ile     #           170     - ttg cag tct cgt tta gct aga aca tca ttt ga - #c aaa aac agg ttc caa      576     Leu Gln Ser Arg Leu Ala Arg Thr Ser Phe As - #p Lys Asn Arg Phe Gln     #       185     - aac att tct gaa aag ctg cac atg gaa tgc aa - #a gca gaa atg gtg acg      624     Asn Ile Ser Glu Lys Leu His Met Glu Cys Ly - #s Ala Glu Met Val Thr     #   200     - cct ctg gtg act aat cct gga cac gtg tgc at - #c acg gac aca aac ctg      672     Pro Leu Val Thr Asn Pro Gly His Val Cys Il - #e Thr Asp Thr Asn Leu     205                 2 - #10                 2 - #15                 2 -     #20     - tat ttt cag ccc ctc aac ggc tac ccg aaa cc - #t gtg gtc cag ata aca      720     Tyr Phe Gln Pro Leu Asn Gly Tyr Pro Lys Pr - #o Val Val Gln Ile Thr     #               235     - ctc caa gat gtc cgc cgc atc tac aaa agg ag - #g cac ggc ctc atg cct      768     Leu Gln Asp Val Arg Arg Ile Tyr Lys Arg Ar - #g His Gly Leu Met Pro     #           250     - ctg ggc ttg gaa gta ttt tgc aca gaa gat ga - #t ctg tgt tcc gac atc      816     Leu Gly Leu Glu Val Phe Cys Thr Glu Asp As - #p Leu Cys Ser Asp Ile     #       265     - tac cta aag ttc tat gaa cct caa gat aga ga - #t gat ctc tat ttt tac      864     Tyr Leu Lys Phe Tyr Glu Pro Gln Asp Arg As - #p Asp Leu Tyr Phe Tyr     #   280     - att gcc aca tac cta gag cac cat gtg gcg ga - #g cac act gct gag agc      912     Ile Ala Thr Tyr Leu Glu His His Val Ala Gl - #u His Thr Ala Glu Ser     285                 2 - #90                 2 - #95                 3 -     #00     - tac atg ctg cag tgg cag cgt gga cac ctt tc - #c aac tat cag tac ctc      960     Tyr Met Leu Gln Trp Gln Arg Gly His Leu Se - #r Asn Tyr Gln Tyr Leu     #               315     - ctt cac ctc aac aac ctg gcc gac cgc agc tg - #c aac gac ctc tcc cag     1008     Leu His Leu Asn Asn Leu Ala Asp Arg Ser Cy - #s Asn Asp Leu Ser Gln     #           330     - tac cct gtg ttt cca tgg ata ata cat gat ta - #t tcc agc tca gaa cta     1056     Tyr Pro Val Phe Pro Trp Ile Ile His Asp Ty - #r Ser Ser Ser Glu Leu     #       345     - gat ttg tca aat cca gga acc ttc cgg gat ct - #c agt aag cca gta ggg     1104     Asp Leu Ser Asn Pro Gly Thr Phe Arg Asp Le - #u Ser Lys Pro Val Gly     #   360     - gcc cta aat aag gaa cgg ctg gag aga cta ct - #g aca cgc tac cag gaa     1152     Ala Leu Asn Lys Glu Arg Leu Glu Arg Leu Le - #u Thr Arg Tyr Gln Glu     365                 3 - #70                 3 - #75                 3 -     #80     - atg cct gaa cca aag ttc atg tat ggg agt ca - #c tac tct tcc ccg ggt     1200     Met Pro Glu Pro Lys Phe Met Tyr Gly Ser Hi - #s Tyr Ser Ser Pro Gly     #               395     - tat gta ctt ttt tat ctt gtt agg att gca cc - #a gag tat atg ctg tgc     1248     Tyr Val Leu Phe Tyr Leu Val Arg Ile Ala Pr - #o Glu Tyr Met Leu Cys     #           410     - ctg cag aat gga aga ttt gat aat gca gat ag - #a atg ttc aac agt att     1296     Leu Gln Asn Gly Arg Phe Asp Asn Ala Asp Ar - #g Met Phe Asn Ser Ile     #       425     - gca gaa act tgg aaa aac tgt ctg gat ggt gc - #a acg gat ttt aaa gag     1344     Ala Glu Thr Trp Lys Asn Cys Leu Asp Gly Al - #a Thr Asp Phe Lys Glu     #   440     - tta att cca gaa ttc tat ggt gat gat gtg ag - #c ttt cta gtc aat agc     1392     Leu Ile Pro Glu Phe Tyr Gly Asp Asp Val Se - #r Phe Leu Val Asn Ser     445                 4 - #50                 4 - #55                 4 -     #60     - ctg aag ttg gat ttg gga aag aga caa gga gg - #a cag atg gtt gac gac     1440     Leu Lys Leu Asp Leu Gly Lys Arg Gln Gly Gl - #y Gln Met Val Asp Asp     #               475     - gtg gag ctt ccc cct tgg gct tcc agt ccc ga - #g gac ttt ctc cag aag     1488     Val Glu Leu Pro Pro Trp Ala Ser Ser Pro Gl - #u Asp Phe Leu Gln Lys     #           490     - agc aaa gat gca ttg gaa agc aat tat gtg tc - #t gaa cac ctt cac gag     1536     Ser Lys Asp Ala Leu Glu Ser Asn Tyr Val Se - #r Glu His Leu His Glu     #       505     - tgg att gat cta ata ttt ggc tac aaa caa aa - #a ggg agt gat gca gtt     1584     Trp Ile Asp Leu Ile Phe Gly Tyr Lys Gln Ly - #s Gly Ser Asp Ala Val     #   520     - ggg gcc cat aat gta ttt cat ccc ctg acc ta - #t gaa gga ggt gta gac     1632     Gly Ala His Asn Val Phe His Pro Leu Thr Ty - #r Glu Gly Gly Val Asp     525                 5 - #30                 5 - #35                 5 -     #40     - ttg aac agc atc cag gat cct gat gag aag gt - #a gcc atg ctt acg caa     1680     Leu Asn Ser Ile Gln Asp Pro Asp Glu Lys Va - #l Ala Met Leu Thr Gln     #               555     - atc ttg gaa ttt ggg cag aca cca aaa caa ct - #a ttt gtg aca cca cat     1728     Ile Leu Glu Phe Gly Gln Thr Pro Lys Gln Le - #u Phe Val Thr Pro His     #           570     - cct cga agg atc acc cca aag ttt aaa agt tt - #g tcc cag acc tcc agt     1776     Pro Arg Arg Ile Thr Pro Lys Phe Lys Ser Le - #u Ser Gln Thr Ser Ser     #       585     - tat aat gct tct atg gca gat tcc cca ggt ga - #a gag tct ttt gaa gac     1824     Tyr Asn Ala Ser Met Ala Asp Ser Pro Gly Gl - #u Glu Ser Phe Glu Asp     #   600     - ctg acc gaa gaa agc aaa aca ctg gcc tgg aa - #t aac atc acc aaa ctg     1872     Leu Thr Glu Glu Ser Lys Thr Leu Ala Trp As - #n Asn Ile Thr Lys Leu     605                 6 - #10                 6 - #15                 6 -     #20     - cag tta cac gag cac tat aaa atc cac aaa ga - #a gca gtt act gga atc     1920     Gln Leu His Glu His Tyr Lys Ile His Lys Gl - #u Ala Val Thr Gly Ile     #               635     - acg gtc tct cgc aat gga tct tca gta ttc ac - #a aca tcc caa gat tcc     1968     Thr Val Ser Arg Asn Gly Ser Ser Val Phe Th - #r Thr Ser Gln Asp Ser     #           650     - acc ttg aag atg ttt tct aaa gaa tca aaa at - #g cta caa aga agt ata     2016     Thr Leu Lys Met Phe Ser Lys Glu Ser Lys Me - #t Leu Gln Arg Ser Ile     #       665     - tca ttt tca aat atg gct tta tcg tct tgt tt - #a ctt tta cca gga gat     2064     Ser Phe Ser Asn Met Ala Leu Ser Ser Cys Le - #u Leu Leu Pro Gly Asp     #   680     - gcc act gtc ata act tct tca tgg gat aat aa - #t gtc tat ttt tat tcc     2112     Ala Thr Val Ile Thr Ser Ser Trp Asp Asn As - #n Val Tyr Phe Tyr Ser     685                 6 - #90                 6 - #95                 7 -     #00     - ata gca ttt gga aga cgc cag gac acg tta at - #g gga cat gat gat gct     2160     Ile Ala Phe Gly Arg Arg Gln Asp Thr Leu Me - #t Gly His Asp Asp Ala     #               715     - gtt agt aag atc tgt tgg cat gac aac agg ct - #a tat tct gca tcg tgg     2208     Val Ser Lys Ile Cys Trp His Asp Asn Arg Le - #u Tyr Ser Ala Ser Trp     #           730     - gac tct aca gtg aag gtg tgg tct ggt gtt cc - #t gca gag atg cca ggc     2256     Asp Ser Thr Val Lys Val Trp Ser Gly Val Pr - #o Ala Glu Met Pro Gly     #       745     - acc aaa aga cac cac ttt gac ttg ctg gcc ga - #g ctg gaa cat gat gtc     2304     Thr Lys Arg His His Phe Asp Leu Leu Ala Gl - #u Leu Glu His Asp Val     #   760     - agt gta gat aca atc agt tta aat gct gca ag - #c aca ctg tta gtt tcc     2352     Ser Val Asp Thr Ile Ser Leu Asn Ala Ala Se - #r Thr Leu Leu Val Ser     765                 7 - #70                 7 - #75                 7 -     #80     - ggc acc aaa gaa ggc aca gtg aat att tgg ga - #c ctc aca acg gcc acc     2400     Gly Thr Lys Glu Gly Thr Val Asn Ile Trp As - #p Leu Thr Thr Ala Thr     #               795     - tta atg cac cag att cca tgc cat tca ggg at - #t gta tgt gac act gct     2448     Leu Met His Gln Ile Pro Cys His Ser Gly Il - #e Val Cys Asp Thr Ala     #           810     - ttt agc cca gat agt cgc cat gtc ctc agc ac - #a gga aca gat ggc tgt     2496     Phe Ser Pro Asp Ser Arg His Val Leu Ser Th - #r Gly Thr Asp Gly Cys     #       825     - ctt aat gtc att gat gtg cag aca gga atg ct - #c atc tcc tcc atg aca     2544     Leu Asn Val Ile Asp Val Gln Thr Gly Met Le - #u Ile Ser Ser Met Thr     #   840     - tca gat gag ccc cag acg tgc ttt gtc tgg ga - #t gga aat tcc gtt tta     2592     Ser Asp Glu Pro Gln Thr Cys Phe Val Trp As - #p Gly Asn Ser Val Leu     845                 8 - #50                 8 - #55                 8 -     #60     - tct ggc agt cag tct ggt gaa ctg ctc gtt tg - #g gac ctc ctt gga gca     2640     Ser Gly Ser Gln Ser Gly Glu Leu Leu Val Tr - #p Asp Leu Leu Gly Ala     #               875     - aaa atc agt gag aga ata cag ggc cac aca gg - #t gct gtg aca tgt ata     2688     Lys Ile Ser Glu Arg Ile Gln Gly His Thr Gl - #y Ala Val Thr Cys Ile     #           890     - tgg atg aat gaa cag tgt agc agt atc atc ac - #a gga ggg gaa gac aga     2736     Trp Met Asn Glu Gln Cys Ser Ser Ile Ile Th - #r Gly Gly Glu Asp Arg     #       905     - caa att ata ttc tgg aaa ttg cag tat taa gt - #gccttttc ctctcctgaa     2786     Gln Ile Ile Phe Trp Lys Leu Gln Tyr     #   915     - tattaaattg aactctattt aatgcatttt taaaccaaac ttttaaacgg ac - #tggtgaat     2846     - gtgcaatgtt agtaattaga agttttacca catggaaaat ttgtggtttt aa - #actttcta     2906     - aatcatggtg acttcattga aagccattag ttgccattct cttagggcag at - #aaaatgcg     2966     - gctgtgttag gaaaaacatg ttacactgta aggcagatga tcgtccccgt at - #gatgattg     3026     - tcagaagaca ggactaagta gcagagaata gctaagagat aaattgggct gg - #ggaaactt     3086     - gtcagaaagc actgaacaat taagaaattt tccaagaaaa tgtgcagtat tc - #tctgctac     3146     - ttctgaatct gttttgtctt cctaatctat cacaattgcc acccatcggg tt - #ttgggtgt     3206     - gtgttttcat agcgtggtta ctttctataa tgctgtaccc agattctaag aa - #cctggaga     3266     - aggattagca gttcttagta agtttactgt gtataggaac ggtttgtatt tc - #attacagc     3326     - tattcatctt ttctacatta aaaatatttt tctctaaaaa aaaaaaaaaa aa - #aa     3380     - <210> SEQ ID NO 2     <211> LENGTH: 23     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Primer     - <400> SEQUENCE: 2     #                23acat gct     - <210> SEQ ID NO 3     <211> LENGTH: 26     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Primer     - <400> SEQUENCE: 3     #              26  aggt actgat     - <210> SEQ ID NO 4     <211> LENGTH: 24     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Probe     - <400> SEQUENCE: 4     #                24acct ttcc     - <210> SEQ ID NO 5     <211> LENGTH: 19     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Primer     - <400> SEQUENCE: 5     # 19               gtc     - <210> SEQ ID NO 6     <211> LENGTH: 20     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Primer     - <400> SEQUENCE: 6     # 20               tttc     - <210> SEQ ID NO 7     <211> LENGTH: 20     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: PCR Probe     - <400> SEQUENCE: 7     # 20               agcc     - <210> SEQ ID NO 8     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 8     #  18              ag     - <210> SEQ ID NO 9     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 9     #  18              aa     - <210> SEQ ID NO 10     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 10     #  18              ct     - <210> SEQ ID NO 11     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 11     #  18              ct     - <210> SEQ ID NO 12     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 12     #  18              ga     - <210> SEQ ID NO 13     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 13     #  18              tg     - <210> SEQ ID NO 14     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 14     #  18              aa     - <210> SEQ ID NO 15     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 15     #  18              gg     - <210> SEQ ID NO 16     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 16     #  18              cc     - <210> SEQ ID NO 17     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 17     #  18              tg     - <210> SEQ ID NO 18     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 18     #  18              ct     - <210> SEQ ID NO 19     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 19     #  18              tc     - <210> SEQ ID NO 20     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 20     #  18              ac     - <210> SEQ ID NO 21     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 21     #  18              ac     - <210> SEQ ID NO 22     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 22     #  18              ta     - <210> SEQ ID NO 23     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 23     #  18              aa     - <210> SEQ ID NO 24     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 24     #  18              at     - <210> SEQ ID NO 25     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 25     #  18              tc     - <210> SEQ ID NO 26     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 26     #  18              gg     - <210> SEQ ID NO 27     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 27     #  18              tg     - <210> SEQ ID NO 28     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 28     #  18              ct     - <210> SEQ ID NO 29     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 29     #  18              ag     - <210> SEQ ID NO 30     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 30     #  18              ct     - <210> SEQ ID NO 31     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 31     #  18              gt     - <210> SEQ ID NO 32     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 32     #  18              tg     - <210> SEQ ID NO 33     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 33     #  18              ca     - <210> SEQ ID NO 34     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 34     #  18              ag     - <210> SEQ ID NO 35     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 35     #  18              tc     - <210> SEQ ID NO 36     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 36     #  18              cc     - <210> SEQ ID NO 37     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 37     #  18              ag     - <210> SEQ ID NO 38     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 38     #  18              gg     - <210> SEQ ID NO 39     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 39     #  18              at     - <210> SEQ ID NO 40     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 40     #  18              tt     - <210> SEQ ID NO 41     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 41     #  18              ct     - <210> SEQ ID NO 42     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 42     #  18              cc     - <210> SEQ ID NO 43     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 43     #  18              tc     - <210> SEQ ID NO 44     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 44     #  18              at     - <210> SEQ ID NO 45     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 45     #  18              ct     - <210> SEQ ID NO 46     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 46     #  18              tt     - <210> SEQ ID NO 47     <211> LENGTH: 18     <212> TYPE: DNA     <213> ORGANISM: Artificial Sequence     <220> FEATURE:     <223> OTHER INFORMATION: Antisense Oligonucleotide     - <400> SEQUENCE: 47     #  18              ag     __________________________________________________________________________ 

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases in length targeted to SEQ ID NO: 1, wherein said antisense compound inhibits the expression of human FAN.
 2. The antisense compound of claim 1 which is an antisense oligonucleotide.
 3. An antisense compound up to 30 nucleobases in length comprising at least an 8-nucleobase portion of SEQ ID NO: 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45 or 47 which inhibits the expression of human FAN.
 4. The antisense compound of claim 3 comprising SEQ ID NO: 8, 9, 10, 13, 17, 18, 19, 20, 21, 22, 23, 27, 29, 30, 31, 33, 34, 35, 36, 37, 39, 41, 43 or
 47. 5. The antisense compound of claim 2 which comprises at least one modified internucleoside linkage.
 6. The antisense compound of claim 5 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 7. The antisense compound of claim 2 which comprises at least one modified sugar moiety.
 8. The antisense compound of claim 7 wherein the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety.
 9. The antisense compound of claim 2 which comprises at least one modified nucleobase.
 10. The antisense compound of claim 9 wherein the modified nucleobase is a 5-methylcytosine.
 11. The antisense compound of claim 1 which is a chimeric oligonucleotide.
 12. A method of inhibiting the expression of human FAN in human cells or tissues in vitro comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of human FAN is inhibited. 